PROJECT SUMMARY/ABSTRACT Cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are used as frontline therapy to treat women with metastatic estrogen receptor positive (ER+) breast cancers given their documented improvements in progression-free and overall survival in this patient population. Furthermore, clinical studies are currently defining their utility in the upfront, non-metastatic setting for women with high-risk ER+ breast cancers. Despite these promising studies, CDK4/6 inhibitors are not yet given in combination with the radiation therapy that patients receive as part of the standard of care. Preliminary data from our lab demonstrates that CDK4/6 inhibition leads to the radiosensitization of multiple ER+ breast cancer cell lines. This radiosensitization occurs to a similar degree with palbociclib, ribociclib, and abemaciclib, the three clinically approved CDK4/6 inhibitors. Although we have demonstrated that all three CDK4/6 inhibitors lead to the radiosensitization of ER+ breast cancer cells, the mechanism of this radiosensitization remains unclear. In this proposal, we aim to 1) determine the mechanism of CDK4/6 inhibitor-mediated radiosensitization of ER+ breast cancer cells and 2) determine the efficacy of CDK4/6 inhibitor-mediated radiosensitization in in vivo models of ER+ breast cancer. Radiosensitization typically occurs through changes in cell cycle distribution or decreases in the efficiency of DNA repair pathways like homologous recombination (HR) or non-homologous end joining (NHEJ). Our lab has preliminary data to suggest that short term CDK4/6 inhibition leads to a decrease in expression of DNA repair proteins like CHK1 and RAD51 that play a role in homologous recombination. CDK4/6 inhibitors halt progression through the G1/S cell cycle checkpoint, and we hypothesize that CDK4/6 inhibitor-mediated G1 cell cycle arrest may limit the ability of cells to undergo DNA double strand break repair through homologous recombination, leading to cell death both in vitro and in vivo. Thus, the overall hypothesis of this work is that pharmacologic CDK4/6 inhibition leads to inhibition of homologous recombination and leads to clinically relevant radiosensitization of ER+ breast cancers in vivo. In Aim 1, we will use reporter assays, immunofluorescence, western blots, qPCR, and cell cycle analysis to determine the effects of CDK4/6 inhibition and radiation on both cell cycle progression as well as HR and NHEJ efficiency in ER+ breast cancer cell lines. In Aim 2, we will use cell line and PDX xenograft models of ER+ breast cancer to assess the efficacy of CDK4/6 inhibitor-mediated radiosensitization in more physiologically relevant systems. The proposed studies will be conducted with guidance from Drs. Corey Speers, James Rae, Ted Lawrence, Lori Pierce, and Daniel Hayes, and will be performed at the University of Michigan. In addition to furthering our understanding of effective treatment strategies for ER+ breast cancer, this proposal advances the National Cancer Institute?s scientific priorities of ?understanding mechanisms of cancer? and ?treating cancer?.